Display technologies have been developed to combine information storage and amplification capabilities of nucleic acids with the functional activities of other compound. Display technologies rely on an association between a functional binding entity (i.e. phenotype) and a nucleic acid sequence informative (genotype) about the structure of the binding entity. Note: Nucleic acid aptamer technology is considered a display technology although a special case as the pheno- and genotype consist of the same molecule (DNA or RNA).
An advantage of such methods is that very large libraries can be constructed and probed for a desired activity of the functional binding entities. Library members having the desired activity can then be partitioned from library members not having the desired activity, thus creating an enriched library with a higher fraction of members having the desired activity. This process is called selection or enrichment. Some display technologies allows for rounds of selections, where the enriched library from one round is amplified and used to prepare a new enriched display library and used in a next round of selection and so forth. The structures of the library members in the enriched library can then be identified by their cognate nucleic acid sequence, thus allowing identification even from minute amounts of material.
Herein relevant libraries may according to the art be termed “in vitro display libraries”.
The term “in vitro display library” shall herein be understood according to the art—i.e. as a library comprising numerous different binding entities wherein each binding entity is attached to a nucleic acid molecule and the nucleic acid molecule comprises specific nucleic acid sequence information allowing to identify the binding entity—i.e. once one knows the specific nucleic acid sequence information of the nucleic acid molecule one directly knows the structure of the specific binding entity attached to the nucleic acid molecule—the structure of the binding entity (i.e. phenotype) attached to the nucleic acid molecule (genotype) is herein termed B-structure.
The prior art describes a number of different methods to make such in vitro display libraries—herein suitable examples include e.g. EP1809743B1 (Vipergen), EP1402024B1 (Nuevolution), EP1423400B1 (David Liu), Nature Chem. Biol. (2009), 5:647-654 (Clark), WO 00/23458 (Harbury), Nature Methods (2006), 3(7), 561-570, 2006 (Miller), Nat. Biotechnol. 2004; 22, 568-574 (Melkko), Nature. (1990); 346(6287), 818-822 (Ellington), or Proc Natl Acad Sci USA (1997). 94 (23): 12297-302 (Roberts), WO06053571A2 (Rasmussen).
As described in e.g. above mentioned prior art—one can today make in vitro display libraries comprising very many (e.g. 1015) specific binding entities (e.g. 1015 different chemical compounds).
In view of this—it is evident that it would be very interesting to be able to improve the selection/enrichment step of such libraries to make an enriched library—e.g. to more efficient be able to identify the structure of a specific binding entity (e.g. a chemical compound) that binds to a target of interest (e.g. a medical important receptor molecule).
In FIG. 3 herein is shown an example of the in vitro display technology as described in EP1809743B1 (Vipergen)—as can be seen in this FIG. 3—the selection step of this example is performed by immobilizing the target (e.g. a receptor) to a solid surface (e.g. a bead or a glass plate).
Without being limited to theory—to our knowledge, the example in FIG. 3 herein may be seen as an example of herein relevant in vitro display technology prior art (e.g. above mentioned prior art)—i.e. to our knowledge the selection for suitable binding entities present within in vitro display libraries are in the prior art generally done by immobilizing the target (e.g. a receptor) to a solid support (e.g. a glass plate, a column, a bead, a nitrocellulose filter, a cell etc) before or after the display library binding event. Non-binders and low affinity binders are typically washed away, whereas the population enriched for binders are recovered from the solid support.
In prior art in vitro compartmentalization (IVC) have been described employed in technologies utilizing phenotype and genotype linkage for interrogating libraries. These prior art technologies can be divided into two groups: a) IVC utilized for facilitating establishing correct phenotype and genotype linkage, which allows for selection of function (e.g. specific target binding) later (post compartment disruption), and b) IVC for facilitating establishing correct phenotype and genotype linkage based on an activity of the phenotype inside the compartment, i.e in a compartment a gene is transcribed and translated and the resulting protein's function inside the compartment is used directly or indirectly for sorting, survival or amplification.
In other words herein relevant so-called IVC prior art technologies—may be described as a:
group a)—wherein the phenotype activity is interrogated AFTER the compartmentalized step; or
group b)—wherein the phenotype activity is interrogated DURING the compartmentalized step.
Examples of IVC prior art belonging to group a):    Bertschinger et al. (2007) Protein Engineering, Design & Selection vol. 20 no. 2 pp. 57-68;    Miller O J, Bernath K, Agresti J J, Amitai G, Kelly B T, Mastrobattista E, Taly V, Magdassi S, Tawfik D S, Griffiths A D. Directed evolution by in vitro compartmentalization. Nat Methods. 2006 July; 3(7):561-70;    Doi, N. and Yanagawa, H. (1999) FEBS Lett., 457, 227-230; and Yonezawa, M., Doi, N., Kawahashi, Y., Higashinakagawa, T. and Yanagawa, H. (2003) Nucleic Acids Res., 31, e118.
Examples of IVC prior art belonging to group b):    Tawfik, D. S. and Griffiths, A. D. (1998) Man-made cell-like compartments for molecular evolution. Nat. Biotechnol., 16, 652-656;    Ghadessy, F. J., Ong, J. L. and Holliger, P. (2001) Proc. Natl Acad. Sci. USA, 98, 4552-4557;    Tay Y, Ho C, Droge P, Ghadessy F J. Selection of bacteriophage lambda integrases with altered recombination specificity by in vitro compartmentalization. Nucleic Acids Res. 2010 March; 38(4):e25. Epub 2009 Dec. 4;    Zheng Y, Roberts R J. Selection of restriction endonucleases using artificial cells. Nucleic Acids Res. 2007; 35(11):e83. Epub 2007;    Mastrobattista E, Taly V, Chanudet E, Treacy P, Kelly B T, Griffiths A D. High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions. Chem Biol. 2005 December; 12(12):1291-300;    Levy M, Griswold K E, Ellington A D. Direct selection of trans-acting ligase ribozymes by in vitro compartmentalization. RNA. 2005 October; 11(10):1555-62. Epub 2005 Aug. 30;    Sepp A, Choo Y. Cell-free selection of zinc finger DNA-binding proteins using in vitro compartmentalization. J Mol Biol. 2005 Nov. 25; 354(2):212-9. Epub 2005 Oct. 3;    Bernath K, Magdassi S, Tawfik D S. Directed evolution of protein inhibitors of DNA-nucleases by in vitro compartmentalization (IVC) and nano-droplet delivery. J Mol Biol. 2005 Feb. 4; 345(5):1015-26. Epub 2004 Dec. 7.
Examples of further IVC prior art may be found in:    Bertschinger et al, (2004) Protein Engineering, Design & Selection vol. 20 no. 2 pp. 699-707;    Chen Yu et al, (November 2008) Nucleic Acid Research, Vol. 36, Nr. 19, Pages: Article No. E128;    Hansen et al. J. Am. Chem. Soc., 2009, 131 (3), pp 1322-1327.